INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 19–1266/2020/23–4–757–762 DOI: 10.17957/IJAB/15.1349 http://www.fspublishers.org Full Length Article Effects of Rotation of Indian Mustard on Cucumber Seedling Rhizosphere Fungal Community Composition Huiting Jia1,2, Muhammad Khashi ur Rahman1,2, Fengzhi Wu1,2 and Xingang Zhou1,2* 1Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture and Rural Affairs, Northeast Agricultural University, Harbin, China 2Department of Horticulture, Northeast Agricultural University, Harbin, China *For correspondence: [email protected]; [email protected] Received 08 August 2019; Accepted 09 December 2019; Published 13 February 2020 Abstract It is well established now that cropping system has great influence on soil microbial communities, but still advance techniques are required to study more of changes in soil microbiota during cropping systems. Here we used high-throughput sequencing to explore the effects of crop rotation with Indian mustard on composition of cucumber rhizosphere fungal community in pot experiment. In our results, an average of 35,748 quality sequences were obtained and these were classified into more than 450 Operational taxonomic units (OTUs) at 97% sequence similarity. The rotation with Indian mustard changed the rhizosphere fungal community composition of cucumber seedlings, but had no effect on fungal community alpha diversity. Rotation with Indian mustard was dominated by Ascomycota phyla, Leotiomycetes and Ascomycota Ineertae sedis classes, orders of Sordariales, Eurotiales, Agaricomycetes Incertae sedis and unclassified Sordariomycetes. In the rotation of Indian mustard, the relative abundance of Humicola, Remersonia, Myrothecium, Scedosporium and Mycothermus spp. was higher, but the relative abundance of Pseudallescheria, Mortierella, Chaetomium, Ilyonectria, Gibellulopsis and Metacordyceps spp. was lower. Overall, this study has provided great insights of changes in fungal community during crop rotation system. © 2020 Friends Science Publishers Keywords: Crop rotation; Cucumber; Fungal community; Indian mustard; Rhizosphere Introduction cucumber rotation with tomato, soybean, wheat and celery was beneficial to maintain the diversity and activity of soil Cucumber is one of the most popular greenhouse microbes and inhibited the harmful microorganisms that vegetables throughout the world. However, continuous were higher in continuously monocropped cucumber monocropping is one of the factor causing “soil rhizosphere (Wu et al. 2011). For example, Jin et al. sickness” which leads to poor plant growth, increase in (2019b) reported that rotation with Indian mustard could soil-borne pathogens and finally reduce crop production suppress cucumber Fusarium wilt disease and increase (Zhou et al. 2017). Soil sickness may be related to plant-beneficial bacteria in rhizosphere. changes in soil microbial communities because of It has been shown that Brassica spp. crops (i.e., autotoxicity (Jin et al. 2020). Previous studies have Indian mustard) are commonly grown to reduce soil- shown that cropping systems, such as rotation, borne pathogenic fungi (Larkin and Griffin 2007) intercropping and interplanting systems, could because when their tissues are disrupted, the significantly improve soil health for better crop glucosinolate releases isothiocyanate, which is toxic to production (Zhou et al. 2017). For example, rotation of many soil pathogenic microorganisms (Motisi et al. tomato-celery-cucumber-Chinese cabbage with 2009). It was found that Indian mustard and wild rocket cucumber could overcome the soil sickness of cucumber green manures increased cucumber rhizosphere bacterial (Zhou et al. 2017). Previous study found that diversity and abundance of potential plant-beneficial incorporation of Brassica juncea inhibit the growth of species, decreased Fusarium wilt disease and enhanced pathogenic Rhizoctonia solani and Fusarium oxysporum expression of defense-related genes in cucumber (Friberg et al. 2009). seedling roots (Jin et al. 2019c). In this study, we Crop rotation is the practice of rotating different crops collected Indian mustard- and the fallow-treated soil sequentially between seasons and years in the same field samples, and further studied the effects of rotation of (Wibberley 1996). Previous studies have shown that Indian mustard on diversity and composition of To cite this paper: Jia H, MKU Rahman, F Wu, X Zhou (2020). Effects of rotation of Indian mustard on cucumber seedling rhizosphere fungal community composition. Intl J Agric Biol 23:757‒762 Jia et al. / Intl J Agric Biol, Vol 23, No 4, 2020 cucumber fungal rhizosphere using high-throughput from each sample in triplicate using PowerSoil DNA sequencing technology. Isolation Kit (MO BIO Laboratories, C.A., U.S.A.) following the manufacturer's instructions. The extracted Materials and Methods DNA (in triplicate) was then combined to make a composite sample and stored at -80°C for further analysis. Greenhouse experiment Illumina miseq sequencing and data processing Cucumber continuous cropping soil was collected from soil upper layer (0–15 cm) of a greenhouse in the experimental As previously mentioned (Zhou et al. 2018a), amplification station (45°41’N, 126°37’E) of Northeast Agricultural of the ITS1 region of the fungal rRNA gene was done using University, Harbin, China, where the cucumber has been the ITS1F/ITS2 primer The forward and reverse primers cultivating since 2006. The soil type used for pot also had a unique 6 bp barcode for each sample. The three experiments was sandy loam and the physicochemical composite sample DNA solutions were separately subjected properties were determined by method as previously used to PCR amplification, then the PCR product was collected by Zhang et al. (2018), which were as follow: EC (1:2.5, and purified and paired-end sequencing (2×300) was w/v) 0.43 mS cm-1; pH 7.64 (1:2.5, w/v); organic matter performed on the Illumina Miseq platform of Majorbio Bio- + - -1 3.51%; inorganic N (NH4 -N and NO3 -N) 146.60 mg kg ; Pharm Technology Co., Ltd., Shanghai, China. Olsen P 284.20 mg kg-1; and available K 341.80 mg kg-1. The de-multiplexing, quality filtering and processing A pot experiment was performed during July to of the raw sequence reads were performed by FLASH September 2016 for cultivation of Indian mustard consisting (Zhou et al. 2017). Identification and removal of chimeric of two treatments in greenhouse (32°C day/22°C night, with sequences was done with USEARCH 6.1 in QIIME a 16 h light/8 h dark and 60–80% relative humidity. Total of (Caporaso et al. 2010). Sequences were classified by the 30 seeds of Indian mustard (cv. Xuelihong) were agglomerative clustering algorithm in USEARCH (Edgar, germinated in each pot (diameter 20 cm, height 17 cm) of 2010) as an Operational taxonomic units (OTUs) with 97% total 10 pots in first treatment (R). Same number of pots sequence similarity. Each representative OTU sequence was without Indian mustard seeds were kept as control treatment then taxonomically classified by BLAST in the Unite (M), and treatments were replicated thrice to make 30 pots database (Koljalg et al. 2013). in total for each treatment. Each pot contained 2.5 ± 0.1 kg of fresh cucumber continuous monocropping soil. After Statistical analysis germination, thinning of seedlings was done to minimize the density of seedlings to 10 by removing bad/extra seedlings To avoid possible deviations due to sequencing depth, a in each pot. Each treatment was replicated thrice to make a random subsample of 30,740 sequences was performed for total of 30 pots in each treatment. Pots of both treatments each sample. The defined OTUs were used to calculate the were placed randomly without any order and their place was taxon cumulative curve. The alpha diversity analysis was changed after every third day. Distilled water was added performed by calculating the Shannon and inverse Simpson every second day to keep soil moisture at about 65% of its indices. The differences in fungal community structures by water content and no fertilizer was applied. Beta diversity analysis were assessed using the UPGMA After 40 days after sowing, the ground portion of hierarchical clustering analysis based on Bray-Curtis Indian mustard was harvested and the underground portion distance. The shared and unique OTUs between treatments was left in the soil. Each pot was wrapped in a black were calculated and their distribution was shown in a Venn polyethylene plastic film, and the soil moisture content diagram. Differences in alpha diversity indices and relative was maintained at around 65% and incubated for 30 days. abundances of microbial taxa between treatments were Cucumber seedlings with two cotyledons (cv. Jinyan 4) analyzed using Student’s t test. All of these analyses were were then planted in pots, one cucumber seedling per pot. done in 'R' (version 3.3.1). The cultivated conditions of cucumber seedlings were same as described above for Indian mustard. Results Soil sampling and DNA extraction Fungal communities alpha and beta diversities After 30 days of plantation, the cucumber rhizosphere After reading and removing a single OTU by basic quality soil was collected according to the method previously control filtration, Illumina Miseq. produced an average of used by Zhou et al. (2017) and sieved through 2 mm 35,748 high quality fungal sequences in each sample with mesh. Sample of 10 plants from